Closed VasLem closed 5 months ago
Hi ! I also see this, in higher depth sequenced targeted gene panels with duplex barcodes.
And it seems strelka chokes on these bases:
[2024-04-07T20:27:56.638567Z] [35329e598677] [23_1] [WorkflowRunner] [ERROR] [2024-04-07T20:27:27.747766Z] [35329e598677] [23_1] [CallGenome+callGenomeSegment_chromId_000_chr1_0007] FATAL_ERROR: Attempting to lookup basecall quality score 89 which exceeds the maximum cached basecall quality score of 70
It seems strelka does not allow bases with quality scores > 70 https://github.com/Illumina/strelka/tree/master/docs/userGuide#alignment-files
This does not happen with CallMolecularConsensusReads.
Is there any way to work around this ?
@VasLem you can determine exactly how many reads are used to build up a consensus base using the per-base SAM tags. You will want to look at the arrays stored in the ac
and bc
tags to see how many reads you've observed per strand. Duplex sequencing further increases accuracy of the final called base by comparing the consensus of both strands and creating a final consensus base call (or No-call if the result is ambiguous).
@karlkashofer you will have to patch Strelka source code yourself and recompile the tool like is done in this PR for manta:
Hi, I have a question regarding CallDuplexConsensusReads. The Phred score for my 5 input samples (which are by the way using a targets and baits targeted sequencing kit, and have about 900 fold enrichment on the targeted regions) is in the range of 35, shown in gray in the plot below. Once the function is called, with default parameters, I am getting transformed reads with very variable scores, shown in purple in the plot. But what concerns me is the huge upscaling of some reads, that end up having a Phred score close to 90. I delved into the maths described here . I understand that the product of probabilities increases the more the reads agree on a single base. For a base with an original Phred score of 35, a Phred score of 90 is translated into a decrease of 5 magnitudes. Does this mean that about 100k reads are on consensus for that base? Thank you in advance!